PROJECT SUMMARY The IgE molecule lies at the center of the pathogenesis of allergic diseases. In sensitized individuals, re-exposure to the offending allergen results in IgE engagement, causing Fc? receptor cross-linking and activation of mast cells and basophils. This triggers the release of mediators into the local tissue, resulting in the vast array of symptoms associated with allergic diseases, including anaphylactic shock. Studies of the human IgE molecule, and its targeted allergens, have been very limited. Nearly all of our knowledge of this process has come from studies using allergic patient serum, which contains a mixture of many antibodies, with many specificities, directed toward many different epitopes, and having many different affinities; thus studies of the molecular interactions of IgE with target allergens are greatly flawed. The ideal way to study this process is to use naturally- occurring human IgE monoclonal antibodies (mAbs), isolated from allergic subjects. Unfortunately, due to many impassible intrinsic technical hurdles no such antibodies have previously ever been made. We have established a method to grow, identify and immortalize IgE encoding B cells by making human hybridomas from the peripheral blood of allergic individuals. In this proposal, we develop the first panel of naturally-occurring alpha- gal-specific human IgE mAbs from subjects with red meat allergy. We intend to define the precise molecular basis for IgE-mediated reactions to red meat, from both the allergen and the antibody perspective. We will begin by generating human hybridomas from peripheral blood B cells of a few highly characterized research subjects. Purified IgE mAbs then will be used to precisely define critical molecular details of human anti-alpha-gal IgE binding. We will employ this panel of antibodies in various molecular assays to assist in localizing, identifying, and ultimately characterizing the allergen(s) found in ticks, responsible for sensitization of humans. As these IgE mAbs represent the B cells which are induced to undergo class-switch recombination, we will use their variable gene sequences to interrogate Illumina sequencing datasets from the research subjects from which the mAbs were obtained. This will allow for a keen understanding of the B cell population(s) underlying this allergic disease and may provide insights needed to predict individuals? risk of sensitization. Together, this proposal will begin studies to define the exact glycoprotein targets of the human IgE B cell response, and the origin of the B cells themselves, which will provide much needed details underlying the sensitization and development of hypersensitivity to this common oligosaccharide.